Polyamide resin-protide conjugate, preparation and uses

ABSTRACT

A large pore polyamide resin is useful for large peptide and protein (protide) synthesis. A method of preparing the same comprises mixing a dimethylacrylamide monomer with an unsaturated or alkenoyl amine monomer, a cross-linker and water, homogeneously emulsifying the aqueous mixture with an organic solvent in the presence of an emulsifier, adjusting the pH of the aqueous mixture during polymerization to 6-8.5 to produce large pore resin beads, and isolating the beads. The beads may be used as a solid phase substrate for the synthesis of a polyamide/protide conjugate. The polyamide resin/protide conjugate may be used, without separation of the protide from the resin or subsequent purification, for immunizing mammals, including humans, against the protide, for affinity purifying immunological molecules binding to the protide, and for immunoassays.

This application is a divisional of 07/693,960, now U.S. Pat. No.5,296,572 filed Apr. 29, 1991 which is a continuation-in-part of U.S.application Ser. No. 07/309,914, now U.S. Pat. No. 5,028,675 filed Feb.10, 1989, by Patrick Kanda, Ronald C. Kennedy and James T. Sparrow,which was a continuation of U.S. application Ser. No. 06/858,216 nowabandoned filed Apr. 30, 1986, by the same inventors entitled POLYAMIDERESIN AND METHOD FOR PREPARATION OF REAGENTS FOR IMMUNODIAGNOSTIC USE.This invention was partially supported by NIH grant HL 27341.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a large pore resin useful for thesynthesis of high MW peptides and proteins (protides) and conjugatesthereof, and to the use of the resin/high MW protide conjugate to inducean immune response in experimental animals. More particularly, thepresent invention relates to a novel large pore polyacrylamide resin, amethod of preparing the polyacrylamide resin, a resin/high MW protideconjugate, a method of preparing a resin/high MW protide conjugate, amethod of inducing immune response in a mammal with the conjugate, and amethod of using the resin/high MW protide conjugate for the affinitypurification of antibodies and for immunodiagnostic purposes.

2. Description of the Background

Solid phase peptide synthesis is a valuable tool for investigating thestructure and mechanism of action of proteins. Most such syntheticmethods involve the use of cross-linked polystyrene resins as the solidphase to which the peptide is anchored during assembly, usually througha linker molecule. Assembly is accomplished by a repetitive cycle ofadding a protected amino acid to the solid phase, selectively removing aprotective group on that amino acid (deprotecting) and adding the nextprotected amino acid.

Although cross-linked, polystyrene resins have been used as supports insolid phase peptide synthesis, their relatively hydrophobic character incomparison to the polar organic media required to solubilize reactantscan be problematic in peptide chain assembly. Such media may freelysolvate a growing peptide, yet incompletely swell the polystyrenematrix. Within the polymer lattice, impaired diffusion of reagents andsteric hindrance can contribute to lowered efficiency during couplingcycles, which, on a repeated basis, lowers final yields appreciably.During the early stages of assembly, when the resin to peptide massratio is high and the physical properties of the support dominate, thislowered efficiency is particularly acute.

Those shortcomings led to the development of a cross-linked,polydimethylacrylamide support which is highly polar in character andfreely permeated by the requisite solvents for peptide synthesis. Suchpolyamide resin, as the aminomethyl derivative, permits the use ofsynthetic schemes incorporating alternate protection strategies throughselection of appropriate linker molecules, which link C-terminalresidues to the support. The thus synthesized peptide or protein,referred to herein as a "protide", may be used in a number ofapplications.

Of particular interest to the present invention is the use of a protideas an immunogen. It has previously been demonstrated that syntheticpeptides analogous to sequences contained in vital-encoded proteins haveproven useful for identifying native antigen determinants associatedwith such proteins, such as various HBsAg synthetic peptides. Theinduction of an antibody response to HBsAg, using such peptides, wasproven to be relatively weak. However, this response could be enhancedthrough coupling of peptides to a carrier protein prior to immunization.

Moreover, the prediction of potential antigenic determinants ofimmunogenic proteins based on primary sequences analysis is far frombeing exact. Because of this, the identification of putative epitopes,through trial and error, may be laborious.

Affinity adsorbents have been prepared using commercially-availableactivated gels such as Bio-Gel P and Bio-Gel A (Bio-Rad), as well asconjugates of Sepharose or Agarose (Pharmacia) with low molecular weightsubstances. Polydimethylacrylamide resins have also been utilized asconjugates with small peptides for the purification of antibodies.

In addition, small peptide/polyamide resin conjugates were synthesizedand utilized for the immunization of experimental animals. Previoustechnology permitted the synthesis on a solid polyamide resin support ofamino acid sequences of limited length. In general, amino acid sequencesof only up to twenty-five or thirty amino acids can be attained byprevious methods.

There is still a need, therefore, for a method of preparing largesynthetic peptides or proteins mimmicking native antigenic sequenceswhich neither requires the purification of the synthetic peptide nor itscoupling to a carrier protein.

SUMMARY OF THE INVENTION

This invention relates to a method of preparing a large pore polyamideresin that comprises

mixing an unsaturated or alkenoyl amine monomer with adimethylacrylamide monomer, a cross-linker and water in a proportion ofmonomer and cross-linker to water of about 1:2 to 1:50 (w/v);

adding an emulsifier in a volume proportion to the aqueous mixture ofabout 1:100 to 1:400;

adding an organic phase to the aqueous mixture;

agitating the aqueous mixture in the presence of the organic phase;

adding an initiator;

adjusting the pH of the aqueous mixture to about 6.0 to 7.5;

adding a promoter to start polymerization to obtain a polyamide resinbead of a pore size capable of lodging a protide of up to about 250,000dalton MW; and

isolating the thus formed polyamide resin bead.

This invention also relates to a large pore polyamide resin prepared bythe method described above. The polyamide resin of the invention has apore size such that it can lodge a protide of up to about 250,000 daltonMW, and in some instances even greater.

Also part of this invention is a method of preparing a polyamideresin/large protide conjugate that comprises

preparing a large pore acrylamide resin by the method of this invention;and

synthesizing an up to about 250,000 dalton MW protide on the resin.

This invention also relates to a polyamide/protide conjugate prepared bythe method of the invention, where the protide may have a molecularweight of up to about 250,000 daltons, and even higher.

Also part of this invention is an immunizing composition that comprises

the polyamide/protide conjugate of this invention; and

a pharmaceutically-accepted diluent.

Also encompassed herein is a method of inducing an immunologicalresponse to a high MW protide in a mammal in need of such treatmentcomprising injecting into the mammal an amount of the resin/protideconjugate of this invention effective to elicit an immunologicalresponse to the protide.

This invention also relates to an in vitro immunoassay method thatcomprises

contacting the resin/protide conjugate of this invention with abiological sample suspected of comprising a molecule having affinity andspecificity for the protide portion of the conjugate;

allowing for the thus defined molecule present in the sample to bind tothe conjugate; and

detecting the presence of any molecule-bound resin/protide conjugate.

This invention also encompasses an immunoassay kit that comprises

the polyamide resin of this invention;

amino acids and other reagents for conducting protide synthesis on theresin; and

anti-human serum.

Also provided herein is an immunoassay kit that comprises

the polyamide resin/protide conjugate described herein; and

anti-human serum.

This invention also relates to a method of purifying a molecule havingaffinity and specificity for a protide from a biological sample, thatcomprises

contacting the resin/protide conjugate of this invention with a samplecomprising a molecule having affinity and specificity for the protideportion of the conjugate;

allowing for any thus defined molecule to bind to the conjugate;

separating the remaining sample from any resin/protide conjugate-boundmolecule; and

separating the resin/protide conjugate from any thus defined molecule.

These and other objects and advantages of the present invention willbecome clear to those skilled in the art from the following description.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

This invention arose from a desire by the inventors to synthesize highmolecular weight polypeptides or proteins (protides) on a solid phasesuch as a polyamide resin. The present invention provides technologythat enables a partitioner to generate various pore size polyamide resinbeads which are capable of lodging high MW protides, e.g., about up to80,000 dalton, and even up to 250,000 dalton MW, and in many instanceseven higher MW.

The resin beads prepared in accordance with this invention havesubstantially large pores which can suitably accommodate protides suchas antibody molecules, e.g., IgG molecules. This makes them extremelyuseful for applications such as affinity chromatography and thepurification of antibody proteins or antipeptides. The resin beadsprepared in accordance with this invention may be utilized to synthesizeprotides of molecular weights of up to 250,000 daltons, and in someinstances even higher molecular weights, which may be cleaved off theresin and separated therefrom. The protide/polyamide resin conjugatesare useful for the immunization of mammals, including humans, as well asfor the production of immunoglobulins, such as IgG. In, addition, theconjugates may also be utilized to purify antibodies from the mammal'sserum or other types of biological samples. Moreover, as alreadyindicated, the polyamide resin/protide conjugate of the invention isalso useful for the purification of any protein having affinity andspecificity for an amino acid sequence contained in the protide, e.g.,antibodies, antipeptides, receptors and the like.

This invention provides a method of preparing a large pore polyamideresin for the solid phase synthesis of high MW protides bypolymerization of a dimethylacrylamide monomer, an unsaturated oralkenoyl amine monomer and a cross-linker in an aqueous medium. In apreferred embodiment, the proportion of unsaturated or alkenoyl aminemonomer, dimethylacrylamide monomer and cross-linker to water is about1:2 to 1:50, and is more preferably about 1:10 to 1:20(w/v). After themonomers, the cross-linker and the water are mixed, an organic phase isadded to the aqueous mixture in a proportion of about 2.5:1.to 3.5:1,and more preferably about 3:1. The aqueous mixture is then agitated inthe presence of the organic phase and an emulsifier in a preferredproportion of aqueous mixture to emulsifier of about 100:1 to 400:1, andmore preferably about 200:1 to 300:1. To start polymerization apolymerization initiator is added. The pH of the aqueous mixture isadjusted to about 6.0 to 7.5, and more preferably to about 6.5 to 7.0. Apromoter is added to obtain polyamide resin beads of a pore size capableof lodging a protide of up to about 250,000 dalton MW. The thus formedbeads of polyamide resin may then be isolated by washing both organicand aqueous solvents away, and used as solid phase for the synthesis ofa protide.

The polyamide resin of the present invention is prepared bycross-linking commercially available dimethylacrylamide monomers inaqueous solution using a cross-linker such as diaminoalkane, preferablya diaminoalkane having alkenoyl groups at either end of the moleculesuch as N,N'-bis-alkenoyldiaminoalkane. In a preferred embodiment of theinvention, the cross-linker is either N,N'-bisacrylyl-1,3-diaminopropaneor N,N'-bisacrylyl-1,4-diaminobutane prepared according to the method ofHalpern and Sparrow (Halpern, J. A., and Sparrow, J. T., SyntheticComm.10:569(1980)), the relevant portion of the text of which isincorporated herein by reference.

The use of the propane analog (n=3) is particularly preferred over theethyl analog because it yields a polymer of larger pore size andimproved swelling properties during protide synthesis. However, it willbe understood by those skilled in the art that other diaminoalkanes,such as N,N'-bisacrylyl-1,2-diaminoethane andN,N'-bisacrylyl-1,6-diaminohexane, are also appropriate for use in thepreparation of the resin of the present invention.

A functional monomer is also included in the synthesis of thecross-linked resin of the present invention. The term "functionalmonomer" refers to those aminoalkenes and N-alkenoyldiaminoalkanes,which are used to anchor the C-terminal amino acid of a syntheticprotide to the resin. The functional monomer may be protected with amethylsulfonylethyloxycarbonyl (MSC) group (Tesser, G. I., andBalvert-Geers, I. C., Int.J. Peptide Protein Res.7:295(1975), in orderto prevent reaction of the free amino group during polymerization. Thefunctional monomers may be prepared by reaction of commerciallyavailable chloride derivatives of MSC with the aminoalkene orN-alkenoyldiaminoalkane to form a urethane. The urethane, in the case ofallylamine, is readily purified, e.g., by passage through silicic acidin chloroform, and crystallized in plates from, e.g., methylenechloride:hexane, and the MSC protective group may subsequently beremoved with base.

The MSC-protected functional monomer is found in general to becompletely soluble in the aqueous solution containing the otherpolymerization monomers. However, the MSC group is not required if thepH is sufficiently acidic to maintain the amino group of the functionalmonomer in a protonated state, i.e., a pH between 6.0 and 7.0.

The amount of functional monomer added is selected to yield a resinsubstitution of between about 0.1 mmol and about 0.7 mmol per gram ofresin, and preferably in the range of about 0.2 to 0.7 mmol/g resin. Forinstance, loadings of about 0.2-0.4 mEq/g resin were realized when thelevel of MSC-allylamine was increased to 20 mole %. The initiator may beany of the initiators known to those skilled in the art such as apersulfate or riboflavin, and is preferably ammonium persulfate.

In a most preferred embodiment, the proportion of cross-linker todimethylacrylamide comprises about 1:4 to 1:50, and more preferablyabout 1:8 to 1:25.

In a preferred embodiment of the present invention, the functionalmonomer is an N-acrylyldiaminoalkane.

The proportion of water added to the monomers and cross-linker iscritical for the formation of large pores in the polymer beads. A higherproportion of water produces larger pores as can be seen from theexemplary disclosure provided below. Typically, the proportion of waterto total amount of monomer and cross-linker is about 2:1 to 50:1, andmore preferably about 5:1 to 20:1.

Because the above-described substances are combined in an aqueousmedium, they are collectively referred to as "the aqueous phase" or"aqueous mixture". The aqueous phase is thereafter added with an organicphase. The term "organic phase" refers to an organic solvent which, whencombined with the aqueous phase and stirred, results in a suspensionfrom which the resin of the present invention may be obtained. In apreferred embodiment, the organic phase comprises a mixture of hexaneand carbon tetrachloride in various proportions. However, other solventsmay also be utilized.

The proportion of aqueous phase to organic phase is typically about1:2.5 to 1:8 by volume, and more preferably about 1:3 to 1:6 by volume.However, other ratios may also be utilized.

An emulsifier is also added. The emulsifier may be any detergent knownto those skilled in the art. In a preferred embodiment, the emulsifiercomprises sorbitan sesquioleate, sorbitan monolaurate, sorbitanmonodecanoate, or mixtures thereof. The amount of emulsifier added isadjusted to give a spherical resin of approximately uniform size.

Typically, the proportion of emulsifier to aqueous phase or mixture isabout 1:100 to 1:400, and more preferably about 1:200 to 1:300 byvolume. A decrease in the amount of detergent below this range mayresult in an emulsion which yields increased amounts of larger,amorphous material, which could contribute to a reduction in the lengthof the internal growing amino acid chains. An increase in the amount ofdetergent above this range may increase the amount of fine material,which is difficult to remove without the loss of significant amounts ofthe resin. Those fines may clog the reaction vessels of the peptidesynthesizer as well as the associated lines and valves.

A solution of promoter titrated to pH between 6.0 and 7.5 may be addedto promote the polymerization of the monomers in suspension, resultingin the formation of beads of the polyamide resin. Alternatively the pHof the aqueous mixture may be adjusted separately as well as thepromoter and initiator added separately. A number of promoters are knownto those skilled in the art, but particular success in preparing thepolyamide resin of the present invention has been obtained withN,N,N',N'-tetramethylethylene-diamine (TEMED).

In a preferred method of preparing the resin, the pH of the promotersolution is adjusted prior to polymerization with dilute mineral acid toabout 6.0, but acceptable resins have been prepared at a pH of about 6.0to about 7.5.

The resulting beads may then be filtered and washed, the MSC group, ifpresent, removed with base, and the beads dried for storage. The beadsmay then be sifted through a mesh or sieve to insure their relativelyuniform size. The Overall yield of the resin prepared using the methodof the present invention may be as high as about 87% to 94% based on thestarting monomers, and in some cases even higher.

The cross-linked polydimethylacrylamide resin of the invention,therefore, takes the following general structure, depending upon theidentity of the functional monomer. ##STR1##

The presently preferred integer is 1 when allylamine is used as thefunctional monomer when A is either --(CH₂)_(y) --NH₂.HCl in the case ofan aminoalkane wherein x is an integer, or --CONH--(CH₂)--_(y) NH₂.HClwhen it is an N-alkenoyl-aminoalkane functional monomer where y is aninteger and 6 when N-acrylyl-1,6-diaminohexane is used as the functionalmonomer and n is an integer. The preferred integer n is either 3 or 4when either N,N'-bisacrylyl-1,3-diaminopropane orN,N'-bisacrylyl-1,4-diaminobutane are used as the cross-linker.

Also provided herein is a conjugate comprising the polyamide resin ofthe invention and an about 800 to 250,000 dalton MW protide. Theconjugate is prepared by a method provided herein. The method comprisespreparing a large pore polyamide resin as described above and thensynthesizing an up to about 250,000 dalton MW protide thereon. Thesynthesis of the protide on the resin is conducted by implementingtechnology known in the art.

The term "protide", as used herein, refers to peptides and proteins ofhigh molecular weight which are synthesized according to the method ofthe present invention. Protides of MW higher than about 20,000 or 30,000dalton MW could not be synthesized previously.

This structure provides maximum exposure of a protide in a polar organicor aqueous solution, and the resin-polymer backbone does not restrictthe protide conformationally. The exposure of the protide is the resultof the ability of the polyamide resin to swell to many times its dry bedvolume when highly solvated by a polar organic solvent or water.

Briefly, a protide is coupled to a polystyrene based resin for solidphase synthesis through a benzyl ester derivative. The separation of thecompleted protide from the resin is usually accomplished by eitheracidic or basic cleavage. Benzyl esters are susceptible to several suchmethods of cleavage, but are also stable throughout the multipledeprotection, neutralization and coupling reactions which arecharacteristic of solid phase synthetic methods. Hydrazine has also beenused to separate the protide from the resin (Kessler, W., and Iselin,S., Helv.Chim.Acta 49:1330(1966)) as have various ammonolytic (Manning,M., J.Am. Chem. Soc.93:1348(1968)), and other methods. These methods,however, require that appropriate steps be taken to avoid damage to theprotide resulting from cleavage followed by purification of the protidefrom the by-products of the synthesis, including amino acids, shortpeptides, decomposition products of the resin, and sometimes, peptidescontaining incompletely removed protecting groups.

Purification may in many instances be accomplished by directcrystallization. Where the contaminating peptides are of a size andcomposition similar to that of the desired product, however, moreselective techniques must be employed. Regardless of the method ofseparation and purification, those requirements add time-consuming stepsto the synthesis, which often lowers the total yield of protide. Themethod of the present invention requires no such separation and/orpurification and, thereby, decreases the length of time required tocomplete the synthesis of the protide while increasing its yield.

A protide may then be synthesized on the beads by direct coupling to theresin or by coupling through a linker which is attached to the resinwith an activator. The term "linker" refers to a linking group whichlinks the carboxyl group of the first amino acid of the protide to thepolymeric resin. In a preferred embodiment, this linker comprises anoxyalkyl benzoic acid (OBA) having one or more amino acid residuescoupled thereto to serve as the first amino acids in the protide chain.Because the OBA linker is used to attach the C-terminal amino acid ofthe first amino acid to the polyamide resin of the present invention,anhydrous hydrogen fluoride may be used to remove the side chainprotecting groups from the protide without significant loss of theprotide from the resin.

In the examples provided below, the first amino acid of choice isglycine, which is protected with a t-butyloxycarbonyl (t-Boc) protectinggroup, but it will be understood by those skilled in the art that theamino acid could be any amino acid, such as the amino acid which is thefirst amino acid in the protide to be synthesized. Other protectinggroups are equally suitable, as well. A short chain-of amino acids mayalso be used which function as a spacer between the protide and theresin-polymer backbone. Polyglycine is one such chain of amino acidswhich may be so used, as well as polyalanine. For synthesis of morecomplex protides, i.e., those which are comprised of multiple chains, orwhen it is desired to synthesize multiple epitopes as may be the casefor synthetic protide antigens, a short chain of amino acids may be usedwhich will accommodate synthesis along two linear sequences. The shortchain of amino acids of choice for such a synthesis, especially when theprotide to be synthesized includes one or more lysine residues, has thefollowing formula.

    u-lys-v

In this formula, u is either (1) the amino acid residue adjacent theN-terminus of lysine in the sequence of the protide to be synthesized,or (2) if it is not desired to synthesize a protide which includes thelysine residue, u is alanine, glycine, or other readily available,preferably charge neutral, amino acid. v is glycine, alanine, or otherelectrically neutral amino acid which is readily commercially available.

In the event it is desired to synthesize multiple epitopes or multiplechain synthetic protides using the short chain of amino acids, synthesisis accomplished through the ε-N of lysine as follows. ##STR2##

In this formula, B is the first synthetic protide to be synthesized, Tis the second synthetic protide to be synthesized, and P is theremainder of the polymerized resin. For example, T and B could representT-cell and B-cell epitopes, respectively, for inducing an immuneresponse in an animal. Those skilled in the art will recognize thatlysine is but one of several amino acids having an amino group in theside chain such that synthesis of the protide T can be accomplished onthe amino acid sidechain. Other such amino acids include ornithine,asparagine, glutamine, arginine, histidine, 5-hydroxylysine, desmosine,isodesmosine, and citrulline. The amino acids lysine, ornithine, and5-hydroxylysine are, however, preferred because of their lack ofelectrical charge, absence of steric hindrance as a result of theirrelatively small side chains, and lack of any stabilizing double bondswhich increase the difficulty of protecting and/or deprotecting the sidechain amino group.

It will also be understood by those skilled in the art that lysine oranother residue having an amino group within the side chain may becoupled directly to the resin or the OBA linker without the u and vamino acid residues. Either way, the resin is effectively double loadedsuch that it is possible to synthesize two protides from the same pointof anchorage to the resin. The resin may also be used to synthesize twoof the same protides simultaneously by, for instance, synthesizing theu-lys-v tripeptide with the Boc protecting group on the ε amino group ofthe lysine residue. The α amino group of the u amino acid may then beprotected with a fluorenylmethyloxycarbonyl (Fmoc) protecting group, theε amino group of the Boc-protected lysine residue is deprotected, and anamino acid substituted onto the ε amino group. The Fmoc protecting groupmay then be removed from the α amino acid, both unprotected amino acidscontacted with the appropriately protected first amino acid in theprotide to be synthesized, and synthesis continued as is known to thoseskilled in the art by repetitive deblocking, selective activation, andaddition of amino acids in the desired sequence.

In the event it is desired to synthesize different protides at the sameanchorage site, it may be necessary to use both Fmoc and Boc protectinggroups. Further, additional protecting groups, stable to both Fmoc-basedand Boc-based synthetic schemes, must be utilized. For instance, if itis desired to add lysine to the chain on which Fmoc-based schemes arebeing used, Fmoc and cbz (on the ε amino group) protecting groups areused, the cbz group being stable to both Fmoc and Boc chemistry.Selection of appropriate protecting groups in this fashion is generallywithin the skill of an artisan.

The method of this invention permits the synthesis of protides of about800 to up to about 80,000 dalton MW, optionally up to about 120,000dalton MW, and in some instances up to about 250,000 dalton MW and evenhigher. By varying the proportion of water and emulsifier to monomersand cross-linker in the aqueous mixture, polymers of different pore sizeare obtained.

Boc-glycyl-4-(oxymethyl) benzoic acid is the preferred linker disclosedherein. It may be prepared by a modification of the method described byMitchell, et al. (Mitchell, et al., J.Org.Chem.43:2845(1978)), theportion of the text which is necessary to enable the method beingincorporated herein by reference. The Mitchell method was modified byeliminating the use of dimethylformamide as a solvent because it isdifficult to evaporate. Even though evaporation may be accelerated byelevating the temperature, the prior art method is still time-consuming.The 4-(bromomethyl)-benzoic acid phenyl ester linker may be converted tothe boc-glycyl derivative by reaction with the tetramethyl-ammonium(TMA) salt of Boc-glycine. ##STR3##

The phenacyl ester may be reductively cleaved with zinc in 85% aceticacid to give Boc-glycyl-(4-oxymethyl) benzoic acid with an overall yieldof about 85% and sometimes higher.

The activators used in the examples to couple the linker to thepolyamide resin prepared as described above are diisopropyl carbodiimideand 4-dimethylaminopyridine. It will, however, be understood by thoseskilled in the art that other activators such asdicyclohexylcarbodiimide (DCC) and 4-methylpyrrolidinopyridine, amongothers, are equally suitable for such a purpose. For instance, theBoc-glycyl-(4-oxymethyl) benzoic acid may be anchored to the aminomethylpolydimethylacrylamide resin using DCC activation with4-dimethylaminopyridine (DMAP) as catalyst.

Also provided herein is a method of preparing a polyamide resin/largeprotide conjugate that comprises

preparing a large pore polyamide resin by the method of this inventionas was described above; and

synthesizing an up to about 250,000 dalton molecular weight protide onthe resin by methods known in the art.

Also provided herein is a polyamide resin/about 800 to 250,000 dalton MWprotide conjugate prepared by the method described above. In a preferredembodiment of the invention, the polyamide resin/protide conjugatecomprises a protide of about 10,000 to 200,000 dalton MW, and morepreferably about 50,000 to 180,000 dalton MW.

An immunizing composition is also provided herein which comprises

the polyamide resin/high MW protide conjugate of this invention; and

a pharmaceutically-acceptable diluent.

The immunizing composition described herein may comprise differentamounts of the conjugate with respect to the diluent. Typically, anamount of polyamide resin/protide conjugate containing about 1.0 to 20mg of the protide will be combined with about 1 ml of diluent, and morepreferably an amount of conjugate comprising 2.0 to 10 mg of protide perml of diluent. Pharmaceutically-acceptable diluents are known in the artand need not be described herein. In a further preferred embodiment, theimmunizing composition further comprises an adjuvant. Suitable adjuvantsare known in the art and will not be described herein. The adjuvant isadded in amounts which are standard in the art.

The polyamide resin/high MW protide conjugate of this invention may beused for a number of purposes, including in vitro assays, inducing animmunogenic response in mammals (immunization) such as humans,purification of immunological molecules having affinity and specificityfor the high MW protide, and the like.

Also provided herein is an in vitro immunoassay, that comprises

contacting the resin/high MW protide conjugate of this invention with abiological sample suspected of comprising a molecule having affinity andspecificity for the protide portion of the conjugate;

allowing for any thus defined molecule present in the sample to bind tothe conjugate; and

detecting the presence of any molecule-bound resin/protide conjugate.

In a preferred embodiment of the invention the method further comprisesbinding or adsorbing the polyamide resin/high MW protide conjugate to asolid support prior to the contacting step. And in still anotherpreferred embodiment, the detecting step comprises radioimmuno-detectionor enzyme/substrate assaying. For instance, an in vitro assay may beconducted by crushing the beaded polyamide resin/high MW protideconjugate with mortar and pestle, and absorbing the crushed conjugateonto a solid phase such as a microtiter test plate with neutral pHbuffer. Serum or other body fluid or biological sample suspected ofcontaining an antibody capable of specifically binding the high MWprotein or peptide bound to the resin may then be incubated with theabsorbed conjugate, unbound antibodies removed by washing, and the boundantibodies detected by an enzyme-linked immunosorbent assay,biotin-avidin amplified assay or other detection methods such asradioimmunoassays which are known in the art.

Also provided herein is an immunoassay kit for practicing theimmunoassay of the invention described above. In one preferredembodiment, the kit comprises

the large pore polyamide resin of this invention;

amino acids and other reagents for conducting protide synthesis on theresin; and

anti-human serum.

In another preferred embodiment the immunoassay kit comprises

the already prepared polyamide resin/high MW protide conjugate of theinvention; and

anti-human serum.

The anti-human serum may be radioactively labeled prior to use or it maybe coupled to a specialized enzyme and used, e.g., as an enzyme-boundimmunoglobulin.

Still another part of this invention relates to a method of purifying amolecule having affinity and specificity for a protide from a biologicalsample, the method comprising

contacting the resin/high MW protide conjugate of the invention with asample comprising a molecule that has affinity and specificity for theprotide portion of the conjugate;

allowing for any thus defined molecule to bind to the conjugate;

separating the remaining sample from any resin/high MW protideconjugate-bound molecule; and

separating the resin/high MW protide conjugate from any thus definedmolecule.

The methods of the invention may be conducted without any separation andpurification steps required by other synthetic methods.

In another embodiment, the conjugate may be crushed and absorbed to asolid support such as a microtiter test plate and the presence ofantibodies in a sample assayed as described above. Separation of theprotide from the resin and purification of the protide is generally notrequired for such an assay.

The polyamide resin/high MW large protide conjugate is also useful as animmunogen. The conjugate may be used directly for immunization with orwithout an adjuvant. For instance, an immune response specific forhepatitis B, as measured by radioimmunoassay, may be induced byimmunization of a mammal, including humans, with a conjugate comprisinga high MW synthetic peptide comprising the amino acid sequence of thehepatitis B surface antigen (HBsAG) peptide 119-159 emulsified inFreund's complete adjuvant. Similarly, immunization may be attained byinjection of a conjugate comprising a high MW peptide corresponding to alarge portion of the protein coat of the AIDS virus HTLV-III and thelarge pore polyamide resin of the invention, a conjugate comprising theinventive resin and a high FEW peptide homologous to a rat fatty acidbinding protein, a large pore resin/high MW peptide conjugate of theinvention comprising a long amino acid sequence homologous to thepredicted sequence of the Abelson murine leukemia virus (ABL), and/or alarge pore polyamide resin/high MW peptide conjugate in which thepeptide is homologous to a high MW protein fragment of apolipoproteins Band/or C.

Immunization may be conducted with varying amounts of the conjugate.Typically, an amount of conjugate comprising about 50 to 200 μg/Kg bodyweight may be given in a single dose, and preferably about 65 to 120μg/Kg body weight. Repeated immunizations may be administered to boostthe effect.

The present invention will be better understood by reference to thefollowing examples, which are presented for purposes of exemplificationonly and are not belied to provide a limitation to the presentinvention.

EXAMPLES Example 1: Preparation of Allylamine Functional Monomer

Five grams (26.8 mmol) 2-methylsulfonyl ethyloxycarbonyl chloride (MSCchloride) (K+K Labs, ICN) were dissolved in 15 ml acetonitrile and addeddropwise over a 20 minute period to a stirred solution of 2.1 ml (28mmol) redistilled allylamine (Kodak) and 4.9 ml (28 mmol) redistilleddiisopropylethylamine (DIEA) in 20 ml acetonitrile. The DIEA (Aldrich)was refluxed over ninhydrin and redistilled. The solution was stirred anadditional 2 hours and the solvent evaporated. The residue was taken upin 250 ml ethyl acetate and allowed to stand for one to two hours. Thebulk of the DIEA hydrochloride salt precipitated as needles. Afterfiltration and evaporation, the crude material was dissolved in aminimal amount of chloroform and loaded onto 60 g of a G-60 silica gelcolumn packed in the same solvent. Elution with chloroform yielded pureMSC-allylamine. R_(F) on TLC=0.64 (Solvent=CHCl₃ : CH₃ OH, 9:1).

The remaining DIEA salts were absorbed to the column under theseconditions. Occasionally, material migrating near the solvent front onTLC contaminated the MSC-allylamine column fractions. That material wasremoved by crystallizing the MSC allylamine from methylenechloride-hexane at -20° C. The yield was 4.8 g (86% from MSC chloride).

Example 2: Preparation of Cross-linker

The cross-linker N,N'-bisacrylyl-1,3-diaminopropane was preparedaccording to the method of Helpern and Sparrow, supra. Briefly,diaminopropane (Aldrich) was dissolved in acetonitrile and addeddropwise to an acrylyl chloride-acetonitrile solution at 4° C., allowedto warm to room temperature and stirred. The diaminopropanedihydrochloride was removed by filtration, washed with warmacetonitrile, and the combined filtrates were concentrated in vacuo.N,N'-bisacrylyl-1,3-diaminopropane was crystallized at 4° C. overnightand the resulting plates filtered and dried in vacuo.

Example 3: Preparation of Polydimethylacrylamide Resin

In a 2-liter cylindrical, fluted polymerization glass vessel fitted witha nitrogen inlet and mechanically driven glass stirrer were added 490 mlhexane and 290 ml carbon tetrachloride. The solution was purged for 15minutes with nitrogen to remove oxygen. An aqueous solution containing2.9 grams (15.9 mmol) N,N'-bisacrylyl-1,3-diaminopropane prepared asdescribed in Example 2 mixed with 18.2 ml (175 mmol)N,N-dimethylacrylamide (PolySciences) were added. 10 g (48 mmol) MSCallylamine prepared as described in Example 1 and 120 ml water wereadded, and the solution was filtered and degassed before addition to theorganic phase. The density of the resulting mixture was adjusted toobtain a uniform suspension with stirring at 400-450 RPM. 0.5 g ammoniumpersulfate (BioRad) in 5 ml H₂ O and 1 ml of either sorbitansesquioleate or sorbitan monolaurate (Sigma) were then added. A solutionof 3 ml N,N,N',N'-tetramethylethylenediamine (TEMED) (BioRad) in 2 ml H₂O, pH 6.5-7.5 (conc. HCl) was then added to the suspension and thesuspended emulsion was stirred for 2 hours under nitrogen atmosphere.

The resulting beaded material was then filtered and washed sequentiallywith water (one liter) methanol (one liter), a mixture ofdioxane:methanol: 2N NaOH (14:5:1, 2 liters, to remove MSC group), water(2 liters), 1N HCl (2 liters), water (2 liters), and then methanol (2liters). The resin was de-fined by suspension in methanol and decanting(3×). After swelling in methylene chloride (Baker HPLC grade), the resinwas shrunk in hexane and dried in vacuo. Large amorphous material wasremoved by sifting the resin through an 80 mesh (180 micron) sieve.

The degree of functionalization was checked by coupling Boc-alanine to100 mg of the resin using diisopropylcarbodiimide as activator and4-dimethylaminopyridine recrystallized from ethyl acetate as catalyst.Amino acid analysis showed a substitution of 0.15 to 0.35 mmol/g resindepending on the lot. Resins were prepared with about 0.1 to 0.5 mmol/gresin depending upon the amount of allylamine added. The loaded resingave no detectable staining with picryl-sulfonic acid, indicating theabsence of unreacted free amine. When swollen in methylene chloride, thebeads occupied about 2.5 times their dry bed volume. When swollen indimethylformamide or an aqueous solution, the beads occupiedapproximately four and six times their dry bed volume, respectively.

Example 4: Preparation of Aminohexyl Resin

A solution of 600 ml of carbon tetrachloride and 1070 ml of hexane wereplaced in a flask and stirred under nitrogen at about 600 rpm. Asolution of 73 g dimethylacrylamide (Poly Sciences), 11.2 gN,N-bisacryloyl-1,2-diaminopropane, and 18.16 gN-acryloyl-1,6-diaminohexane HCl in 500 ml cold water were added to theorganic solution. The density adjusted by adding hexane or carbontetrachloride until the aqueous solution stayed suspended when thestirrer was stopped. 2 g ammonium persulfate dissolved in 2 ml waterwere then added, followed by about 1 ml of sorbitan laurate and asolution of 6 ml of TEMED in 6 ml of water pH 6.0 (12N HCl).Polymerization was allowed to proceed for about 3 hours, after whichtime the stirrer was stopped, hexane was added, the resin allowed tosettle, and the organics removed by aspiration.

The resin was transferred to a 3 liter filter funnel, washed inmethanol, water, and methanol, resuspended in methanol, and allowed tosettle, and fine material was removed from the methanol by aspiration.The resin was washed with 3×2 l dimethylformamide and methylenechloride, stirred and then washed with ethylacetate and hexane. Theresin was then dried in a vacuum dessicator and passed through an ASTM80 mesh screen to obtain about 93 grams of resin. After neutralizingwith 100 ml of 1N sodium hydroxide per gram of resin, washing withwater, methanol, and methylene chloride, the loading was determined byamino acid analysis to be 0.7 mEq/g after coupling Boc-alanine to theresin.

Example 5: Preparation of Linker

The linker Boc-glycyl-4-(oxymethyl) benzoic acid was prepared by amodification of the method of Mitchell, et al., supra. Briefly,4-(bromomethyl) benzoic acid phenylacylester was prepared by dissolving10.3 ml redistilled diisopropylethylamine and 12.05 g (60.6 mmol)bromoacetophenone in 450 ml ethyl acetate. 13.89 g (60.6 mmol)4-(bromomethyl) benzoic acid were added in seven equal portions over a 3hour period to the stirred solution at 40°-50° C. Stirring was continuedfor 2 more hours at room temperature. Precipitated Et₃ N.HBr was removedby filtration and the ethyl acetate solution was washed four times with50 ml each of an aqueous solution of 10% citric acid, saturated sodiumchloride, saturated sodium bicarbonate, and saturated sodium chloride.The organic phase was dried over anhydrous magnesium sulfate and freedof solvent by rotary evaporation under reduced pressure. The residue wascrystallized from CH₂ Cl₂ -petroleum ether (1:3 v/v) to give the4-(bromomethyl) benzoic acid phenacylester.

4-(bromomethyl) benzoic acid phenylacylester was converted toBoc-glycyl-4-(oxymethyl) benzoic acid by dissolving 25 mmol (4.38 g)Boc-L-glycine in 15 ml methanol and titrating to neutrality with 25%tetramethyl-ammonium hydroxide in methanol. The solvent was removedazeotropically with chloroform in vacuo, and the salt was dissolved in150 ml acetonitrile. To the stirred solution were added 5.8 g (17.5mmol) 4-(bromomethyl) benzoic acid phenacyl ester prepared as described.After overnight mixing, the precipitated tetramethylammonium bromide wasfiltered and the solvent evaporated. The residue was dissolved in 400 mlethyl acetate and the solution filtered.

The organic phase was then washed successively with 10% aqueous citricacid (3×75 ml), 0.5M sodium bicarbonate:0.5M potassium carbonate (2:1)pH 9.5 (8×75 ml), and then water (3×75 ml). The solution was dried withMgSO₄, and the solvent removed in vacuo. The residue was dissolved in200 ml 85% acetic acid to which 23 g acid washed zinc dust were added.The mixture was stirred until the phenacyl ester was no longer visibleby TLC, about 4 to 5 hours. The zinc dust was filtered out and washedwith 50 ml acetic acid, and the combined solutions were lyophilized. Theresidue was suspended in 100 ml water:300 ml ethyl acetate, and the pHadjusted to 1.5 with concentrated HCl. The aqueous layer was extractedwith a second portion of 200 ml ethyl acetate and the combined extractswere washed with 100 ml water. After drying with MgSO₄ and evaporating,the Boc-glycyl-4(oxymethyl) benzoic acid was purified byrecrystallization from methylene chloride:hexane at -10°. The yield was4.5 g (14.5 mmol, 83% from the phenacyl ester).

Example 6: Coupling of Linker to Polyamide Resin

Boc-glycyl-4-(oxymethyl) benzoic acid prepared as described in Example 5was coupled to 1.2 g aminomethyl polyamide resin prepared as describedin Example 3, as well as the aminohexyl resin prepared as described inExample 4, on a Biosearch Sam II automated peptide synthesizer usingdiisopropylcarbodiimide and dimethylaminopyridine as activator in a 1:1methylene chloride:dimethylformamide solution. Both methylene chloride(Baker HPLC grade) and dimethylformamide (Baker Photrex grade) were usedwithout further purification.

Following treatment with hydrogen fluoride, 50 mg of the glycyl resinprepared as described in Example 3 were found to contain 0.13 mmol/g oflinker by amino acid analysis. Amino acid analysis was performed using aBeckman Model 119 amino acid analyzer following either 2 hour hydrolysis(12N HCl:propionic acid, 1:1, 135° C.) or 24 hour hydrolysis (6N HCl,110° C.) of resin-bound peptides.

Example 7: Synthesis of Hepatitis B Antigen Peptide

The hepatitis B surface antigen (HBsAg) peptide 119-159 was assembled onthe aminomethyl, cross-linked polydimethylacrylamide resin prepared asdescribed in Example 3, having the Boc-glycyl-4-(oxymethyl) benzoic acidlinker prepared as described in Example 5 attached thereto using themethod described in Example 6, with all residues being double coupledusing a Biosearch Sam II automated peptide synthesizer. The sequence ofthat HBsAg peptide is as follows relative to the AYW subtype. ##STR4##

The peptide included the following substitutions to control the specificformation of disulfide loops. Serines substituted for cysteines 121,138, and 149. The cysteines 139 and 147 sulfhydryls were blocked by the4-methoxybenzyl group, while the sulfhydryls of the cysteines at 124 and137 were protected as the S-acetamidomethyl derivatives. α-N-tBocprotected amino acids were purchased from Bachem. Additional side chainprotecting groups were as follows: formyl group for the indole nitrogenof tryptophan; benzylethers for threonine and serine hydroxyls;acetamidomethyl or 4-methoxybenzyl for cysteine sulfhydryls as describedabove; benzyl esters for β-carboxyl of aspartic acid and the γ-carboxylof glutamic acid; 2-chlorobenzyloxycarbonyl for ε-amino group of lysine;2,6-dichlorobenzyl ether for the phenolic hydroxyl of tyrosine; and thep-tosyl group for the guanidine of arginine. For the synthesis,methylene chloride (Baker HPLC grade) and DMF (Baker Photrex grade) wereused without further purification. Diisopropylethylamine (DIEA)(Aldrich) was refluxed over ninhydrin and redistilled. Trifluoroaceticacid (Halocarbon) was redistilled, with the middle cut used indeblocking steps. All other chemicals were reagent grade or better andused without further purification.

Side chain protecting groups were removed from the completedpeptidyl-resin by treatment with anhydrous HF (20 ml/g resin) at 0° for30 minutes, containing 10% anisole and 2% ethanedithiol. Followingevaporation of HF, the peptidyl-resin was washed successively withether, 1% acetic acid, methanol, 5% DIEA in methylene chloride,methanol, then water. The peptidyl-resin was dried in vacuo. The formylgroup was removed from the tryptophan by treatment with 1M ethanolamineat 0°. A disulfide bridge was formed between cysteines 139 and 147 bypotassium ferricyanide treatment. A second disulfide bridge betweencysteines 124 and 137 resulted during simultaneous removal of theacetamidomethyl moieties with a solution of iodine in acetic acid.

Example 8: In Vitro Assay for Presence of HBsAg Antibody

Human serum is assayed for the presence of antibody specific for theHBsAg peptide 119-159 by the following in vitro assay. A quantity of theHBsAg peptide 119-159-polyamide resin conjugate prepared as described inExample 6 was crushed with mortar and pestle. A microscope was used toverify that the polyamide resin-peptide conjugate was crushed.Approximately 100 μl of a solution containing about 200 nanograms to 10micrograms of crushed polyamide resin-peptide conjugate in a neutral pHbuffer such as phosphate buffered saline (PBS) was absorbed to a solidphase such as Dynatech Immunolon II Microtiter test plates. Nonspecificbinding sites were blocked with 10% normal goat serum (NGtS) and theplate was washed with Tween 20 PBS (T-PBS) to remove unbound antibodies.

Human sera suspected of containing antibodies specific for HBsAg peptide119-159 and rabbit antisera produced by immunizing rabbits with thepolyamide resin-HBsAg peptide 119-159 conjugate diluted in 10% NGtS werethen added to the polyamide resin-peptide-coated plate and incubated for1 hour at 37° C., followed by washing with T-PBS. Biotin goat anti-humanIgG or biotin goat anti-rabbit IgG (Vector Laboratories, Burlingame,Calif.) was then incubated with the bound human and rabbit sera,respectively, for 1 hour at 37° C. The wells were washed and avidinconjugated to horseradish peroxidase (Av-HRP) was added for 20 minutesat room temperature. The wells were then washed with T-PBS to remove anyunbound Av-HRP, and peroxidase activity was determined using a 1 mMsolution of 1,2'-azino-di(3-ethylbenzthiazoline sulfonic acid) (SigmaChemical Co.) and 0.03% H₂ O₂ as substrate. The reaction was stoppedwith 5% (w/v) sodium dodecyl sulfate in water prior tospectrophotometric quantitation at 410 nm using a Dynatech plate reader.Optimal dilutions of each reagent were selected by titration. Allreagents for determining specific binding except the substrate werediluted in 10% NGtS.

Example 9: In Vitro Assay for Presence of HBsAg Antibody

Human serum was assayed for the presence of antibody to hepatitis Bsurface antigen by the following in vitro assay. A 10% solution of thepolyamide resin-HBsAg peptide 119-159 conjugate was prepared in abuffered bovine serum albumin (BSA) solution containing a finalconcentration of 40% tetrahydrofuran. An equal volume of antibodyspecific for the HBsAg peptide 119-159 containing about 100,000 to1,000,000 counts per minute I¹²⁵ were added and incubated with gentlerocking. The resulting suspension was centrifuged and the pellet washedwith 1% BSA-Tween 20 PBS, and then centrifuged again. The radioactivityof the pellet was then counted in a Gamma counter. The results clearlyindicate the recognition of the polyamide resin-HbsAg peptide 119-159conjugate by native HBsAg antibody.

                  TABLE 1    ______________________________________    In Vitro Assay of HBsAg Antibody                  Glycyl resin                             Resin-HBsAg peptide    Sample        (Control)  119-159 conjugate    ______________________________________    IgG Human anti-HB1                  1240 cpm*  22,840    IgG Human anti-HB2                  1921       28,732    Normal human IgG                  1432       1,949    ______________________________________     *all measurements in counts per minute

Example 10: Use of Polyamide Resin Protide Conjugate to Induce anImmunogenic Response in Mammals

The polyamide resin-peptide conjugate prepared as described in Example 7was used to induce an immunogenic response in rabbits as follows. NewZealand white female rabbits were immunized with three monthlyintramuscular injections of either 200 μg HBsAg peptide 119-159 as thepeptide/resin conjugate, or only glycyl-resin emulsified in Freund'scomplete adjuvant. A range of immunogen of about 50 μg to 1 mg was usedfor rabbits. Serum was collected after bi-weekly bleeding and checkedfor anti-HBsAg activity using a commercially available radioimmunoassay(RIA) kit (AUSAB, Abbott Laboratories). The recognition of the nativeHBsAg surface antigen by the anti-peptide 119-159 antibody responseinduced in the rabbits is demonstrated by the following data developedby that RIA.

                  TABLE 2    ______________________________________    Immunization with Different Immunizers                                Antibody    Rabbit                      Titer.sup.a    Immunogen       Immunization                                (RIA units/ml)    ______________________________________    1    Glycine-Resin  Preimmune.sup.a                                    ≦8.sup.b                        Primary     ≦8                        Secondary   ≦8                        Tertiary    ≦8    2    HBsAg Peptide-Resin                        Preimmune   ≦8                        Primary     ≦8                        Secondary   183                        Tertiary    920    3    HBsAg Peptide-Resin                        Preimmune   ≦8                        Primary     ≦8                        Secondary   72                        Tertiary    262    ______________________________________     .sup.a Sera obtained prior to immunization.     .sup.b Antibody titer to HBsAg is below the sensitivity of the RIA kit an     is considered not to contain specific antibodies.

As can be seen from these data, the polyamide resin-HBsAg peptide119-159 conjugate containing a single disulfide bridge between cysteines139 and 147, when used to immunize rabbits, yieldes anti-peptideantisera which cross reactes with HBsAg.

Example 11: Synthesis of HTLV-III Antigen Peptide

The amino acid sequence between residue numbers 503-532 of the gp120HTLV-III envelope glycoprotein was assembled on the cross-linkedpolydimethylacrylamide resin prepared as described in Example 3 wherethe Boc-glycyl-4-(oxymethyl) benzoic acid linker was prepared asdescribed in Example 5 and attached thereto using the method of Example6, in the same method as described for the synthesis of the HBsAGpeptide 119-159 in Example 7, the only difference being the order inwhich the protected amino acids were added. The sequence of amino acids503-532 of the gp120 HTLV-III peptide is as follows. ##STR5##

Using the same method, three additional peptide-resin conjugates havebeen prepared which include the following amino acid sequences which arehomologous to portions of the envelope glycoprotein of HTLV-III, eachidentified by their respective residue numbers.

                  TABLE 3    ______________________________________    Sequencer Utilized    Sequence     Homologous to    ______________________________________    304-327      gp120    341-370      gp120    733-756      gp41    ______________________________________

Example 12: Ability of HTLV-III Peptide/Resin Conjugates to Bind toHuman anti-HIV Antibodies

HTLV-III is one of several strains of retrovirus which have beenimplicated as the causative agent of human Acquired ImmunodeficiencySyndrome, or AIDS, all of which are referred to as HumanImmunodeficiency Virus, or HIV. To test the immunogenicity of thepeptide/resin conjugate of the present invention, 4 conjugates havingpeptides homologous to portions of the HTLV-III envelope glycoproteinprepared as described in Example 11 were tested against serum samplestaken from 110 HIV-infected humans for their ability to bind anti-HIVantibodies. That assay was conducted as follows.

2 μg samples of each conjugate were absorbed to Dynatech Immunolonmicrotiter test plates in borate buffered saline (BBS) pH 8.2 overnightat 4° C.

Non-specific sites were blocked with 200 μl 10% normal goat serum (NGtS)in Tween 20-phosphate buffered saline (T-PBS) for 20 minutes at roomtemperature and then washed three times with T-PBS. Biotin goatanti-human IgG (Vector Laboratories, Burlingame, Calif.) was thenincubated with the bound human sera for 1 hour at 37° C. Wells werewashed 3 times and avidin conjugated to horseradish peroxidase (Av-HRP)was added for 20 minutes at room temperature. The wells were againwashed 3 times with T-PBS and peroxidase activity determined using a 1mMsolution of 1,2'-azino-di(ethyl-benzthiazolinesulfonic acid) (SigmaChemical Co.) with 0.03% H₂ O₂ as the substrate. The reaction wasstopped with 5% (w/v) sodium dodecyl sulfate in water and opticaldensity read at 410 nm using a Dynatech plate reader. Optimal dilutionswere selected by titration.

The results were as shown in Table 4 below. A positive result wasdefined as an optical density three standard deviations above the meanoptical density obtained with 20 seronegative samples.

                  TABLE 4    ______________________________________    Peptide Sequences Used that Bound h-antibodies    Peptide Sequence Individuals                               Positive    ______________________________________    304-327          26        24    341-370          33        30    503-532          44        40    733-756          24        22    ______________________________________

Example 13: Use of Polyamide Resin/HTLV III Synthetic Peptide ConjugateTo Induce an Immunogenic Response

Rabbits immunized with the polyamide resin/HTLV-III peptide 503-532conjugate produced a specific anti-peptide response as determined by anenzyme linked immunosorbent assay conducted according to the method ofExamples 8 with the use of antisera produced by immunization of rabbitswith the polyamide resin/HTLV-III peptide 503-532 conjugate rather thanthe conjugate including the HBsAg peptide 119-159 used in that Example,and in Example 12. The results of the immunoassay are presentedgraphically in FIG. 1. The data represented by the circles is data fromrabbits immunized with that conjugate, the data represented by trianglesis from those same rabbits before immunization. The solid circles andtriangles are from one rabbit and the open circles and triangles arefrom a second rabbit. A conjugate comprised of the polyamide resin and athird peptide failed to demonstrate significant binding. One of therabbits produced an anti-HTLV-III response specific for the HTLV-IIIgp120 envelope protein based on a radioimmunoprecipitation assayconducted according to the method of Allan and Barin (Allan, J. S., etal., Science 228:1091 (1985); Barin, F., et al., Science 228:1094(1985)), portion of which are incorporated herein in their entirety byreference to the extent necessary for enablement.

The ability of rabbit antisera against peptide 503-532 to neutralizeHTLV-III infectivity was assessed on the basis of a reduction of reversetranscriptase activity using a tenfold dilution of the HTLV-III stockwith a constant amount of antisera (Barre-Sinoussi, F., et al., Science220:868 (1983)). A single rabbit anti-peptide 503-532 antiserumefficiently reduced HTLV-III replication at day 10 compared to pooledhuman sera from AIDS patients at tenfold dilutions of virus. A secondrabbit antiserum to that peptide failed to reduce HTLV-III replicationand so was used as a control throughout the RT assay. No anti-HTLV-IIIactivity was detected in this particular antiserum based onradioimmunoprecipitation even though the rabbit received a similarimmunogen and produced a detectable anti-peptide response.

The antiserum that neutralized HTLV-III detected both gp120 and gp160envelope glycoproteins. This rabbit antiserum was found to be lessefficient in neutralizing HTLV-III compared to human AIDS serum on day12 and 15 following HTLV-III infection.

Example 14; Production of Antibodies Using Peptide/Resin Conjugates

To further test the immunogenicity of the peptide-resin conjugates ofthe present invention, 4 additional peptide-resin conjugates wereprepared, injected into rabbits, and their sera tested for the presenceof antibody against the respective native proteins. Those peptide-resinconjugates were prepared in the same manner as set out in Example 7, andincluded the following peptides.

(a) A peptide having a sequence homologous to a portion of the aminoacid sequence of the rat fatty acid binding protein residue numbers22-34 (FABP) (Chan, L., et al., J. Biol. Chem. 260:2629 (1985)).

(b) A peptide having a sequence homologous to a portion of the predictedamino acid sequence of the murine Abelson leukemia virus (abl 389-403;Reddy, E., et at., Proc. Nat'l. Acad. Sci. USA 80:3617(1983)).

(c) A peptide having a sequence homologous to a portion of the aminoacid sequence of the apolipoprotein C-II, residue numbers 56-69 (ApoC-II) (Hospattanker, A. V., et al., J. Biol. Chem. 260:318 (1984)).

(d) a peptide having a sequence homologous to a portion of the aminoacid sequence of the apolipo protein B, residue numbers 3357-3369 (ApoB) (Yang, C.-Y., et al., Nature 323:738 (1986)).

Peptides were coupled directly to the aminohexyl resin prepared asdescribed in Example 4 containing 0.70 mM glycine per gram resin forsynthesis. Once synthesis was completed, the resins were dried and 0.5 gof each resin sample was treated with 10 ml anhydrous HF containing 10%anisole and 1% ethanedithiol for 30 minutes at 0° C. HF was evaporatedunder vacuum at 0° C. and the resin transferred to a glass filter funnelwith ether, washed with 3×50 ml each of ether, methanol, water, 1%acetic acid, water, 0.1M Tris, water, 1% acetic acid and water toneutrality, and then dried in a vacuum dessicator. The amino acidcomposition of each sample was determined by hydrolysis with 12NHCl:propionic acid (1:1) at 135° C. for 2 hours and analyzed on aBeckman 6300 amino acid analyzer. Amino acid sequences of each peptidewere determined with either an Applied Biosystems Model 470A or 477Aprotein sequencer.

Samples of each peptidyl-resin (1 mg) were swollen in 100 μl of sterilenormal saline and crushed with an homogenizer. After mixing with anequal volume of incomplete Freund's adjuvant, the mixtures were injectedinto New Zealand white rabbits at multiple sites. After 2 weeks, serumwas obtained from an ear vein and checked with an ELISA test forantibody against the native protein. Rabbits were then boosted withmixtures prepared in this same manner and the process repeated over twomonths. Antibody titers after 2 months, determined against nativeproteins (not the respective peptide) were as shown in Table 5 below.

                  TABLE 5    ______________________________________    Antibody Production    Peptide      Antibody Titer    ______________________________________    FABP         900    ABL-389      64    Apo C-II     250    Apo B        50    ______________________________________

The experiment was repeated after synthesizing a T-cell epitope fromStaph. aureus nuclease having the following amino acid sequence.

    lys-met-val-glu-asp-ala-lys

The T-cell epitope was added synthetically to the amino terminal of theremaining peptidyl-resin conjugate for each of the peptides, viz., FABP,ABL-389, Apo C-II, and Apo B. The remaining steps were repeated exactlyas above. The following antibody titers were found.

                  TABLE 6    ______________________________________    Antibody Titers    Peptide      Antibody Titer    ______________________________________    FABP         2500    ABL-389      2550    Apo C-II     1250    Apo B         250    ______________________________________

In a second set of tests, the ABL-resin and FABP resin conjugates wereground in a tight-fit homogenizer with 20 strokes, and 500 μg of theground conjugate was suspended in 1.5 ml of phosphate buffered saline(PBS) and emulsified with another 1.5 ml of Freund's complete adjuvant.The rabbits were injected subcutaneously at multiple sites, and twobooster injections of each PBS-conjugate suspension in Freund'sincomplete adjuvant were given at 2 week intervals. Two weeks after thesecond booster, the rabbits were bled and anti-peptide titer assayed inan ELISA test against the respective peptide of the resin-peptideconjugate.

The ELISA test was conducted by plating 50 ng of each peptide in 0.1MNaHCO₃ pH 9 in the wells of a 96-well test plate and drying overnight at37° C. Non-specific sites were blocked with a TN solution of 50 mMTris-HCl pH 7.6 and 50 mM NaCl with 5% non-fat dry milk for 1 hour at37° C. 50 μl sera were added in serial dilution to each well andincubated at 37° C. for 1 hour. The plates were washed 6 times with TNsolution containing 0.05% Tween 10 (Sigma). 50 μl ofperoxidase-conjugated goat anti-rabbit IgG (1:4000) (Boehringer MannheimBiochemicals) in TN buffer with 5% nonfat dry milk were added to eachwell, and after incubating for 1 hour, plates were washed six times withTN solution. 50 μl of o-phenylene-diamine (OPD) (Sigma) were added toeach well as substrate and incubated for 20 minutes at room temperature.

The plates were read at 495 nm in an ELISA autoreader as a net value inwhich the optical density (OD) reading of the pre-immune sera wassubtracted from the OD of the immune sera. Optical density is plotted asa function of serum concentration in FIG. 2. Each data point representsthe average of three rabbits. The circles represent the data fromrabbits immunized with the ABL-resin conjugate and the trianglerepresents data from rabbits immunized with the FABP-resin conjugate.

Example 15: Preparation of Aminohexyl Polydimethylacrylamide Resin withPores Accepting 40,000 Dalton MW Proteins

Polydimethylacrylamide resin beads containing an aminohexyl group wasprepared as follows in a siliconized 3-liter indented, cylindricalpolymerization glass vessel (Reliance Glass) fitted with a nitrogeninlet and mechanically drived siliconized glass propeller-type stirrer.554 ml of hexane and 346 ml of carbon tetrachloride were added to thevessel. The solution was purged for 15 min with nitrogen to removeoxygen. A cold aqueous solution containing 2.5 g (13.7 mmol)N,N'-bisacrylyl-1,3-diaminopropane, 18.2 g (184 mmol)N,N-dimethyl-acrylamide, 4.54 g (22 mmol) N-acrylyl-1,6-diaminohexanehydrochloride monomers and 250 ml cold water was prepared. This solutionwas filtered and degassed and then added to the organic phase. Thedensity of the aqueous/organic mixture was adjusted by addition ofhexane or carbon tetrachloride to obtain a uniform suspension of theaqueous phase. 0.5 g Ammonium persulfate in 1 ml H₂ O and 1.6 mlsorbitan monolaurate were added while stirring at 400-450 rpm. Asolution of 1.5 ml N,N,N',N'-tetramethylethylenediamine (TEMED) in 1.5ml H₂ O pH 6.5 (conc. HCl) was then added after ensuring an appropriatedroplet size.

Polymerization commenced after about 30 to 45 minutes and stirring wascontinued for 3 to 4 hours under a nitrogen atmosphere. Hexane was thenadded, stirring stopped and the beaded resin allowed to settle. Thesupernate was removed with suction and the beaded material filtered on a3 l siliconized glass funnel and washed sequentially with methanol (2×1l), water (2×1 l) and methanol (2×1 l). The resin was de-fined bysuspension in 1 l methanol, allowed to settle and any suspended materialremoved by decantation.

This process was repeated 3 times. Washing was concluded with methylenechloride (3×1.5 l), ethyl acetate (1×1.5 l) and hexane (2×1 l), and theresin was then dried in vacuo. Any large amorphous material was removedby sifting the resin through an 80 mesh (180 micron) sieve. Overallyields were up to about 90 to 99%.

The degree of functionalization was confirmed by neutralizing 3 g ofaminohexyl hydrochloride resin with 1N NaOH and then washing with water,methanol, and methylene chloride. Boc-glycine was coupled as thesymmetric anhydride formed with dicyclohexylcarbodiimide and catalyzedwith 4-dimethylaminopyridine recrystallized from ethyl acetate. Aminoacid analysis showed a substitution of about 0.5 to 0.7 mmol/g resin,depending on the lot. The loaded resin gave no detectable staining withpicrylsulfonic acid, indicating the absence of unreacted free amine.

The pore size of the Boc-glycyl resin was determined by washing 3 g ofthe resin on a siliconized funnel with methanol, water, and 0.01M sodiumphosphate buffer pH 7.4, transferring to a siliconized filter flask,degassing and placing it in a 0.9×28 cm column. Mixtures of peptides andproteins of known molecular weights were then loaded onto the column andeluted with phosphate buffer. The elution profile and the log molecularweight v. elution volume are not shown but were obtained. From theseprofiles, the pore size was estimated to be large enough to accommodatea protein of molecular weight 40,000 Daltons.

Example 16: Preparation of Aminohexyl Polydimethylacrylamide Resin withPores Accepting 80,000 Dalton MW Proteins

A resin was prepared as above except that the monomers were dissolved in375 ml cold water and suspended in 433 ml of carbon tetrachloride and693 ml hexane at a stirring speed of 495 rpm. 0.5 g ammonium persulfatein 1 ml water and then 1.2 ml sorbitan monolaurate were added to obtaina desirable droplet size. The polymerization was allowed to proceed for3.5 hours, and the beaded resin was then washed as above and loaded inthe same manner with Boc-glycine. The loading was 0.63 mmol/g.

After washing and preparing a column of the resin, the pore size of theBoc-glycyl resin was determined in the manner described above. Theelution profile and the log molecular weight v. elution volume are notshown but were obtained. The pore size of the resin was estimatedtherefrom to be large enough to accommodate up to about an 80,000 DaltonMW protein.

Example 17; Preparation of Aminohexyl Polydimethylacrylamide Resin withPores Accepting 120,000 Dalton MW Proteins

A resin was prepared as above except that the monomers were dissolved in500 ml of cold water and suspended in 519 ml of carbon tetrachloride and831 ml hexane at a stirring speed of 570 rpm. 0.5 g ammonium persulfatein 1 ml of water, and then 1.3 ml sorbitan monolaurate were added toobtain a desirable droplet size. The polymerization was allowed toproceed for about 3.5 hours, and the beaded resin was washed as aboveand loaded in the same manner with Boc-glycine. The loading was 0.27mmol/g.

After washing and preparing a column of the resin, the pore size of theBoc-glycyl resin was determined in the manner described above. Theelution and the log molecular weight v. elution volume profiles are notshown but indicate that a protein of up to 120,000 Daltons MW can beaccommodated by the resin.

Example 18: Preparation of Aminohexyl Polydimethylacrylamide Resin withPores Accepting 250,000 Dalton MW Proteins

A resin was prepared as above except that the monomers were dissolved in500 ml cold water and suspended in 519 ml carbon tetrachloride and 831ml hexane with stirring at 500 rpm. 0.5 g ammonium persulfate in 1 ml ofwater, and then 1.4 ml sorbitan monolaurate were added to obtain anappropriate droplet size. After the polymerization was allowed toproceed for 3.5 hours, the beaded resin was washed as above and loadedin the same manner with Boc-glycine. The loading was 0.42 mmol/g.

After washing and preparing a column of the resin, the pore size of theBoc-glycyl resin was determined in the manner described above. Theelution profile and the log molecular weight v. elution volume plot (notshown) indicate a protein of 250,000 Daltons MW can enter the pores ofthe resin.

Example 19: Preparation of Rabbit-antibodies against WT NuclearTransport Signal Peptide

A synthetic peptide was conjugated to keyhole limpet hemocyanin (KLH)using the heterobifunctional crosslinker MBS. The conjugate wasrepeatedly injected into rabbits as an emulsion containing Freund'sincomplete adjuvant. Serum was obtained after the 4th. and 5th.injections. IgG was prepared by removal of albumin by caprylic acidprecipitation, followed by ammonium sulfate precipitation of IgG. Theprecipitated IgG was resuspended in PBS and dialyzed.

Example 20: Immunopurification of Rabbit Anti-WT Nucelear TransportSignal Peptide on a Peptide Bound Resin

0.12 mg of peptide resin were swollen in phosphate buffer saline (PBS)that contains 100 uM leupeptin and 1 mM PMSF.

The resin was packed into a small column and the column washed with 25ml of PBS at a flow rate of 15 ml per hour. 27.5 mg of IgG in 13 mlrabbit anti-peptide IgG were applied to the column and circulatedthrough the column overnight with the aid of a peristaltic pump. Thecolumn was washed with 25 ml PBS, 25 ml PBS+1M NaCl, and then with 25 mlPBS.

The bound IgG was slowly eluted with 15 ml of ammonium thiocyanate. Each1 ml fractions were collected and the optical density (OD) of eachfraction at 280 nm was determined. Peak fractions were then dialyzedagainst PBS.

The amount of starting IgG, unbound IgG, and affinity purified IgG weredetermined by an ELISA assay after layering them on wells previouslycoated with BSA-SPDP-WT peptide or keyhole limpet hemocyanin (KLH).

Example 21: ELISA Assay

Microtiter wells were incubated with 200 ng of BSA-SPDP-peptide or KLH,incubated overnight in PBS, and then blocked with PBS containing 10% NGSfor 30 minutes at 37° C. The microtiter wells were washed 3 times withPBS containing 0.3% Tween and then incubated for 1 hour at 37° C. withrabbit anti-peptide IgG at different concentrations in PBS containing10% NGS. The wells were washed 3 times with PBS containing 0.3% Tweenand then incubated for 1 hour at 37° C. with goat anti-rabbit IgGconjugated with horse radish peroxidase diluted in PBS containing 10%NGS. The wells were washed 3 times with PBS containing 0.3% Tween andincubated with a peroxidase substrate 2,2'-azino-bis(3-ethylbenzthiazoline) sulfonic acid for 30 minutes at 23° C. Theoptical density of the samples was read at 410 nm.

                  TABLE 7    ______________________________________    ELISA Results            ##STR6##                         Unbound or  Affinity    ug IgG/well             Start       Flow Through                                     Purified    ______________________________________    125 ug              ##STR7##                          ##STR8##                                      ##STR9##    31.3              ##STR10##                          ##STR11##                                      ##STR12##    7.8              ##STR13##                          ##STR14##                                      ##STR15##    1.9              ##STR16##                          ##STR17##                                      ##STR18##    ______________________________________

Each row represents the results of successive passages of the unbound,or flow-through fractions through the column, resulting in progressivelygreater binding specificity of the fractions for the peptide vs. KLH.

The invention now being fully described, it will be apparent to one ofordinary skill in the art that many changes and modifications can bemade thereto without departing from the spirit or scope of the inventionas set forth herein.

We claim:
 1. A method of preparing a resin/large MW protide conjugate,comprisingpreparing a large pore polyamide resin by mixing anunsaturated or alkenoyl amine monomer with a dimethylacrylamide monomer,a cross-linker and water in a proportion of monomer and cross-linker towater of about 1:2 to 1:50 (w/v); adding an emulsifier in a proportionto the aqueous mixture of about 1:100 to 1:400, adding an organic phaseto the aqueous mixture; agitating the aqueous mixture in the presence ofthe organic phase, adding an initiator; adjusting the pH of the aqueousmixture to about 6.0 to 7.5; adding a promoter to start polymerizationto obtain polyamide resin beads of a pore size capable of lodging aprotide of up to about 250,000 dalton MW; isolating the thus formedpolyamide resin beads; and synthesizing an up to about 250 Kdalton MWprotide on the resin.
 2. A polyamide protide conjugate prepared by themethod of claim
 1. 3. An composition, comprisingthe polyamide/protideconjugate of claim 2; and a diluent.
 4. The immunizing composition ofclaim 3, further comprisingan adjuvant.
 5. An immunoassay kit,comprisinga large pore polyamide resin prepared by mixing an unsaturatedor alkenoyl amine monomer with a dimethylacrylamide monomer, across-linker and water in a proportion of monomer and cross-linker towater of about 1:2 to 1:50 (w/v), adding an emulsifier in a proportionto the aqueous mixture of about 1:100 to 1:400, adding an organic phaseto the aqueous mixture, agitating the aqueous mixture in the presence ofthe organic phase, adding an initiator, adjusting the pH of the aqueousmixture to about 6.0 to 7.5, adding a promoter to start polymerizationto obtain polyamide resin beads of a pore size capable of lodging aprotide of up to about 250,000 dalton MW, and isolating the thus formedpolyamide resin beads; amino acids and other reagents for conductingpeptide synthesis on the resin; and anti-human serum.
 6. An immunoassaykit, comprisingthe polyamide resin/protide conjugate of claim 2; andanti-human serum.
 7. A method of purifying a molecule having affinityand specificity for a protide from a biological sample,comprisingcontacting the resin/protide conjugate of claim 1 with asample comprising a molecule having affinity and specificity for theprotide portion of the conjugate; allowing for any thus defined moleculeto bind to the conjugate; separating the remaining sample from anyresin/protide conjugate-bound molecule; and separating the resin/protideconjugate from any thus defined molecule.
 8. The method of claim 7,whereinthe molecule comprises an antibody.
 9. The method of claim 1,wherein the alkenoylamine monomer comprises an allylamine monomer. 10.The method of claim 9, wherein the allylamine monomer comprises anN-alkenoyl-diaminoalkane.
 11. The method of claims 1, wherein thecrosslinker comprises a diaminolkane.
 12. The method of claim 11,wherein the diaminoalkane comprises a N,N'-bisalkenoyl-diaminoalkane.13. The method of claim 12, wherein the N,N'-bisalkenoyl-diaminoalkaneis selected from the group consistingofN,N'-bisalkenoyl-1,3-diaminopropane;N,N'-bisalkenoyl-1,3-diaminoethane; N,N'-bisalkenoyl-1,3-diaminobutane;and N,N'-bisalkenoyl-1,3-diaminohexane.
 14. The method of claim 1,wherein the allylamine monomer carries a protective group.
 15. Themethod of claim 14, further comprising removing the allylamineprotective group from the polyamide resin by contacting the beads with abase.
 16. The method of claim 14, wherein the allylamine protectivegroup comprises methysulfonylethyloxycarbonyl.
 17. The method of claim1, whereinthe organic phase comprises a mixture of hexane and carbontetrachloride; and the emulsifier is selected from the group consistingof sorbitan monolaureate, sorbitan decanoate, and sorbitan sesquiolate.18. The method of claim 1, wherein the beads are isolated by filtering,washing and drying.
 19. The method of claim 1, further comprisingcoupling a linker to the polyamide resin beads.
 20. The method of claim1, wherein the proportion of cross-linker to dimethylacrylamidecomprises about 1:4 to 1:50.
 21. The composition of claim 3, wherein thediluent comprises a pharmaceutically-acceptable diluent.